PROJECT SUMMARY Pore-forming toxins (PFTs) are the most common type of cytotoxic proteins produced by bacterial pathogens. At high levels, these toxins can lyse target host cell membranes, but during an actual in vivo infection it is likely rare that PFTs reach lytic concentrations. Instead, recent work from our lab and others indicates that sub-lytic amounts of PFTs can affect host cell functions in more indirect ways by modulating host signaling and proteolytic cascades. The PFT ?-hemolysin (HlyA) is often expressed by strains of Extraintestinal Pathogenic Escherichia coli (ExPEC), the leading cause of both bloodstream and urinary tract infections (UTIs). During an infection, HlyA can inactivate host phagocytes, alter inflammatory responses, and disrupt tissue barriers. Consequently, ExPEC strains that express HlyA are able to cause more severe and disseminated infections. The mechanisms by which HlyA and related PFTs alter host functions and elicit tissue damage without causing outright host cell lysis are poorly understood. Previously, we demonstrated that HlyA intoxication triggers the cellular redistribution and activation of TRY4. HlyA-induced activation of TRY4 in bladder epithelial cells and macrophages leads to the proteolytic cleavage of numerous host components, including several cytoskeleton- associated factors and central regulators of host inflammatory responses. Interestingly, others have shown that the aberrant activation and extracellular release of TRY4 can stimulate the metastatic growth of several cancer cell types and may also promote the development of neurodegenerative disorders like Alzheimer's disease and multiple sclerosis. The pathways by which TRY4 is activated and re-localized in these diseases, or in response to HlyA intoxication, are for the most part unknown. Here, we propose to define the mechanisms and consequences of HlyA-induced TRY4 activation and re-localization using cell culture and murine models of UTI and sepsis. This work will provide fresh insight into the functions of a widespread ExPEC-associated toxin, and will likely be relevant to HlyA- like PFTs that are encoded by a number of other important bacterial pathogens. In addition, our results may aid the development of new anti-virulence strategies for combating ExPEC and could help explain the dysregulation of TRY4 that occurs with other pathological conditions that are not typically associated with infectious disease.